Brain Research, 526 (1990) 181-185 181 Elsevier BRES 15785 Research Reports CRF-like effects of sauvagine and urotensin I on synaptosomal tyrosine hydroxylase activity of mouse striatum Pierluigi Onali and Maria C. Olianas Department ofNeurosciences, University of Cagliari, Cagliari (Italy) (Accepted 27 February 1990) Key words: Sauvagine; Urotensin I; Corticotropin-releasing factor receptor; Tyrosine hydroxylase; Mouse striatum In the present study we have investigated whether sauvagine (SVG) and urotensin I (UT), two peptides displaying sequence homology with corticotropin-releasing factor (CRF), could affect synaptosomal tyrosine hydroxylase (TH) activity of mouse striatum in a manner similar to CRF. The enzyme activity was assayed in supernatants obtained following sonication and centrifugation of homogenates preincubated with the peptides. SVG and UT produced a concentration-dependent increase of TH activity with a half-maximal effect obtained at 5 and 10 nM, respectively. SVG and UT were as effective as CRF with maximal stimulations corresponding to 52-58% increase of basal enzyme activity, whereas the rank order of potency was SVG > UT = CRE Kinetic analysis of TH activity versus low concentrations of the pterin co-factor (0.05-0.4 mM) indicated that the stimulations elicited by CRF, SVG and UT were associated with an increase in the V,~ax of the enzyme form with high affinity for the co-factor. The CRF receptor antagonist a-helical CRF9_41 inhibited the effects of all 3 peptides. Moreover, the combined addition of CRF with either SVG or UT did not produce additive effects on TH activity. The stimulatory effects of CRF, SVG and UT were dependent on the concentration of extracellular free Ca2÷, being minimal in a Ca2+-free medium and maximal at about 0.5 mM extracellular free Caz÷. These results indicate that SVG and UT can mimic the effect of CRF on synaptosomal TH by acting on a common receptor site associated with a Ca2+-dependent mechanism. INTRODUCTION Originally identified as a hypophysiotropic peptide that stimulates the release of ACTH and fl-endorphin from the pituitary 28, corticotropin-releasing factor (CRF) has subsequently been found to affect a variety of brain functions independently of its action on the pituitary. When injected into ventricular or parenchymal sites of rat brain, CRF increases neuronal discharge rates 7'29, in- duces behavioral activation and increased emotionality 15, inhibits food intake 19 and sexual activity27 and enhances sympathetic outflow 4. At a biochemical level, the central administration of CRF alters catecholamine metabolism in different brain areas 6'14. Recently, we have observed that, in vitro, CRF is a potent stimulator of synaptosomal dopamine (DA) synthesis in rat and mouse striatum 2°. This effect is associated with a stable activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. Thus, an increased catecholaminergic function may mediate some of the central actions of CRE Sauvagine (SVG) and urotensin I (UT), two peptides isolated from the frog skin and from the teleost urophysis, respectively 9,16, display sequence homologies with CRE Moreover, SVG and UT have been found to mimic the effects of CRF on pituitary function 23, animal behavior 2'18, food intake 2'11 and sympathetic activity3. In the present study we investigated whether the two peptides share with CRF the ability to stimulate synap- tosomal TH activity. MATERIALS AND METHODS Materials L-[1-14C]tyrosine (55.3 mCi/mmol), [aa2-p]ATP (30-40 Ci/mmoi) and [2,8-3H]cyclic AMP (25 Ci/mmol) were obtained from Dupont- New England Nuclear. Synthetic human/rat CRF and a-helical CRF9_41 were from either Peninsula Labs (Belmont, CA, U.S.A.) or Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sauvagine, urotensin I and all the other reagents were from Sigma. Angiotensin II, neurotensin and substance P were from Peninsula. L-Aromatic amino acid decarboxylase was prepared according to Waymire et al.3°. Measurement of TH activity Male Swiss albino mice (30-40 g) were killed by cervical translocation and striata were rapidly dissected on an ice-cold plate. The tissue was immediately immersed in ice-cold and freshly-made 0.32 M sucrose and a 10% (w/v) homogenate was prepared at 4 °C using a teflon/glass tissue grinder (clearance 0.25 mm, Kontes, Vineland, NJ, U.S.A.) at 800 rpm with two cycles of 6 up-and-down strokes. In most experiments the homogenate was used as a synaptosomal source. When the Ca2÷ dependence of TH activation was studied, a crude mitochondriai fraction (P2) was prepared as described by Gray and Whittakerm. TH activity was assayed in supernatant obtained following sonication and centrifugation of the tissue preparation preincubated with the peptides. Routinely, the Correspondence: P. Onali, Department of Neurosciences, via Porcell 4, 1-09124 Cagliari, Italy. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)